Studies on the cytoplasmic protein tyrosine kinase activity of the Antarctic psychrotrophic bacterium Pseudomonas syringae

Studies on the cytoplasmic protein tyrosine kinase activity of theAntarctic psychrotrophic bacterium Pseudomonas syringae

Pratik Jagtap, M.K. Ray *Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India

Received 1 October 1998; received in revised form 28 December 1998; accepted 10 February 1999

Abstract

The Antarctic psychrotrophic bacterium Pseudomonas syringae contains a 66-kDa cytoplasmic protein which was found tobe phosphorylated on a tyrosine residue [Ray, M.K. et al. (1994) FEMS Microbiol. Lett. 122, pp. 49^54]. To investigate thenature of the cytoplasmic protein tyrosine kinase and its role in the bacterial physiology, we carried out some biochemicalstudies of the enzyme in vitro in the presence of exogenous peptide substrates and expression studies in vivo at low and hightemperature during various phases of growth. The results suggest that the protein tyrosine kinase associated with thecytoplasmic fraction of the bacterium has certain similarities and dissimilarities with the known eukaryotic tyrosine kinases.The protein tyrosine kinase could phosphorylate exogenous substrate corresponding to the N-terminal peptide of p34cdc2

kinase but could not do so on poly(Glu:Tyr). The enzyme could not be inhibited by genistein, staurosporine and dimethylaminopurine, but could be inhibited by piceatannol which is a known competitive inhibitor of the peptide binding site ofmammalian protein tyrosine kinases. The enzyme activity in the cytoplasm is uniquely inhibited by sodium orthovanadate(IC50=20 WM) which is a known protein tyrosine phosphatase inhibitor. The expression studies show that the enzyme isproduced more at a higher temperature (22³C) of growth than at lower temperature (4³C) and during the stationary phase ofgrowth of P. syringae. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Allrights reserved.

Keywords: Antarctic psychrotroph; Pseudomonas syringae ; Protein tyrosine kinase activity

1. Introduction

Protein phosphorylation on a tyrosine residue hasnow been shown to be present in all three domainsof life, namely Eukarya, Archaea, and Bacteria [1^3].This post-biosynthetic modi¢cation of protein wasoriginally discovered in higher vertebrates and wasfound to play an important role in various signal

transduction cascades which are crucial to physiolog-ical and developmental processes of the organisms[3]. However, the importance of tyrosine phospho-rylation in bacteria is still not clearly understood.In pathogenic bacteria, such as enteropathogenic Es-cherichia coli (EPEC), the role of tyrosine phos-phorylated protein (BipA) in host-pathogen interac-tions has been advocated [4]. However, very littleprogress has been made in elucidating the catalyticproperties of bacterial protein tyrosine kinases (Ptks)and their substrate speci¢cities. Only recently, one

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 0 5 - 6

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* Corresponding author. Tel. : +91 (40) 7172241;Fax: +91 (40) 71711957; E-mail: [email protected]

FEMS Microbiology Letters 173 (1999) 379^388

membrane associated protein from Acinetobacterjohnsonii [5] and a cytoplasmic protein from Esche-richia coli [6] have been puri¢ed, which have beenshown to possess an autophosphorylating Ptk activ-ity.

Antarctic psychrophilic and psychrotrophic bacte-ria constitute a very useful model system to study themechanisms of cold adaptation [7^10]. One of ourmajor interests in the area of cold adaptation liesin understanding how the Antarctic psychrotrophicbacteria, which grow in a temperature range of 0^30³C, sense environmental temperatures and how thesensing process is coupled to the transcription appa-ratus of the bacteria. Although the bacteria in gen-eral use a `two component' regulatory system con-sisting of the sensor kinases and response regulatorsfor sensing environmental cues [11^13], no such sys-tem has been demonstrated for sensing temperatures.Our attempt to identify such a system in the Antarc-tic bacteria led us to identify some speci¢c membraneproteins which are di¡erentially phosphorylated atlow and high temperatures in the Antarctic psychro-trophic bacterium Pseudomonas syringae [14]. Duringthis study we also identi¢ed a cytoplasmic 66-kDaprotein which was phosphorylated on tyrosine resi-dues [15]. The phosphorylation of the protein in-creased in the presence of a membrane fraction athigher temperatures (22^30³C) in vitro. The signi¢-cance of this observation, however, was not clear.Therefore, it was of interest to study the biochemicalproperties of the Ptk which is present in the cyto-plasm of P. syringae and also the production of theenzyme during various growth phases of the psy-chrotrophic bacterium with a view to obtain a clueto the nature of the enzyme and its physiologicalimportance. Here we report the results of such astudy which re£ects the nature of the tyrosine kinaseof the psychrotrophic bacterium P. syringae.

2. Materials and methods

2.1. Bacterial strains and growth conditions

The Antarctic psychrotrophic bacterium P. syrin-gae Lz4W which grew optimally at 22³C was iso-lated, identi¢ed and maintained as reported earlier[16]. Routinely, the culture was grown on ABM

(Antarctic bacterial medium, which contains pep-tone, 0.5% (w/v) and yeast extract, 0.2% (w/v)) atroom temperature (22³C) or at cold temperature(4³C) when needed. In experiments wherein the e¡ectof vanadate on growth was studied, varying concen-trations of sodium orthovanadate (100, 125 and250 WM) were added to the medium. The growthof the organism was monitored by measuring theoptical density (OD) at 600 nm. For studies on thegrowth phase-dependent tyrosine kinase activity, thecells were grown at both low (4³C) and high (22³C)temperatures and harvested at di¡erent OD600 val-ues.

2.2. Isolation of bacterial cytosolic proteins

The bacterial cytosolic extract was prepared asdescribed earlier [14] following a method similar toSchnaitman [17]. Brie£y, the cells were treated withlysozyme (60 Wg ml31� in a membrane bu¡er (10 mMTris, pH 8.0, 0.75 M sucrose, 2 mM EDTA and 1 WMPMSF) and sonicated for 2 min in a Branson soni-¢er. The unbroken cells were separated by centrifu-gation at 8000 rpm for 10 min in a Sorvall centri-fuge. The total protein was separated into amembrane fraction and a cytosolic fraction by ultracentrifugation at 30 000 rpm for 2 h at 4³C in a TL-100 table-top ultracentrifuge (Beckman). The super-natant was used as the cytosolic fraction in thisstudy. For some of the experiments, the cytosolicfraction was subjected to further ammonium sulfatefractionation. The 80^100% ammonium sulfate frac-tion was enriched with the tyrosine kinase activity.

2.3. Assay of Ptk activity

The cytoplasmic Ptk activity was assayed by usingtwo di¡erent methods, namely a radioactive assayusing [Q-32P]ATP and a non-radioactive ELISAbased assay using anti-phosphotyrosine antibody.The radioactive assay in vitro was carried out usingthe cytosolic fraction as described earlier [14].Brie£y, 40^50 Wg of the cytosolic proteins were in-cubated in the presence of [Q-32P]ATP (10 WCi, 3000Ci mmol31) in 10 mM Tris-HCl (pH 7.5) and 5 mMMgCl2 for 10 min at 0 or 22³C. After 10 min, anequal volume of 2Usample bu¡er was added, boiledfor 2 min, cooled and analysed on a SDS-polyacryl-

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amide gel [18]. The phosphorylated proteins werevisualised either by autoradiography or by using aphosphorimager (Molecular Dynamics). For quanti-¢cation, the autoradiograms were scanned by a den-sitometer (Molecular Dynamics) and the phosphor-images were analysed by software provided with thephosphorimager. It is to be noted that the valuesobtained by either methods were compatible and,in general, proportionate to the amount of radioac-tivity incorporated in the bands.

The ELISA-based Ptk activity in the cytosolicfraction was measured by a non-radioactive tyrosinekinase assay kit (Boehringer Mannheim) followingthe supplier's protocol. Two biotin-labelled sub-strates, PKS1, (Biotin-KVEKIGEGTYGVVYK-amide) corresponding to the amino acids 6^20 ofthe cell division kinase p34cdc2 [19], and PKS2, cor-responding to the amino acid sequence 1^17 of gas-trin (Biotin-EGPWLEEEEEAYG WMDF-amide)[20], were used in the experiments. Brie£y, 10 pmolof the biotin-labelled substrates were incubated inthe presence of cytosolic extract (40^100 Wg of pro-tein) and ATP (1 mM). The reactions were carriedout at 30³C for 30 min. The peptide substrates(PKS1 and PKS2) which were phosphorylated bythe Ptk in cell extracts were immobilised in the strep-tavidin-coated wells of a microtitre plate and de-tected by peroxidase conjugated anti-phosphotyro-sine antibody. The colour which develops due tothe antigen-antibody complex and a peroxidase sub-strate (provided with the kit) is measured spectro-photometrically at 405 nm (reference wavelength490 nm) on an ELISA plate reader (Molecular De-vices). The calculation was based on the di¡erence ofthe absorbance at 405 and 490 nm, as speci¢ed bythe supplier's protocol. The speci¢c activity of theenzyme was expressed as pmol of phosphotyrosinemin31 (mg of protein)31.

The following inhibitors were used in some experi-ments: staurosporine (1^20 WM), genistein (10^50WM), sodium orthovanadate (1^300 WM), okadaicacid (0.5^2 WM), sodium £uoride (50^200 mM), di-methyl amino purine (1^10 mM). The e¡ects of var-ious divalent cations (MgCl2, MnCl2 and CaCl2)were also studied in the in vitro assays at the e¡ec-tive concentrations of 5, 10 and 50 mM each. As apositive and negative control for these inhibitors to-wards mammalian Ptks and protein tyrosine phos-

phatase (Ptpase) studies were carried out with ratliver extract and hamster reproductive tissues([20a], K. Uma Devi and S. Shivaji, unpublishedobservation).

2.4. Other analytical methods

Proteins were measured by a modi¢ed method ofthe Lowry procedure [21]. The SDS-PAGE analysisof proteins was carried out by the method ofLaemmli [18]. The phospho-amino acids were ana-lysed by TLC of the acid hydrolysates of the 32P-labelled proteins following an incubation in 6 NHCl for 6 h at 120³C [22] as described earlier [15].

3. Results

3.1. The Ptk of P. syringae could phosphorylate asynthetic peptide substrate

An earlier identi¢cation of the cytosolic tyrosinephosphorylated 66-kDa protein in the cytoplasm ofthe Antarctic psychrotrophic P. syringae indicated

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Fig. 1. Cytosolic Ptk activity of the Antarctic P. syringae. Theassay was carried out in the presence of cytosol (100 Wg of pro-tein) and the exogenous peptide substrate PKS 1 by a ELISAbased method. The values are the average of two experimentsonly.

P. Jagtap, M.K. Ray / FEMS Microbiology Letters 173 (1999) 379^388 381

the existence of a Ptk in the bacterium [15]. To learnmore about the kinase, it was necessary to ¢nd anexogenous peptide substrate suitable for biochemicalanalysis of the enzyme. Poly(Glu:Tyr), a routinelyused substrate for mammalian tyrosine kinase, couldnot be phosphorylated by the cytoplasmic fractionwhich could phosphorylate the 66-kDa protein.However, the substrate PKS 1 corresponding to theamino acid 6^20 of the cell division kinase (p34cdc2)[19] was phosphorylated by the cytoplasmic fractionof P. syringae to a modest degree in a concentration-dependent manner and the apparent Km for PKS 1was 10 WM (Fig. 1). The substrate PKS 2 corre-sponding to the amino acids 1^17 of gastrin [20]also could be phosphorylated by the kinase but toa lesser extent (data not shown). The cause for thissubstrate discrimination could not be ascertained inthis study.

3.2. Inhibitors of the Ptk of P. syringae

To examine the nature of similarities and dissim-

ilarities between the Ptks of P. syringae and higherorganisms, various inhibitors of the eukaryotic Ptksand Ptpase were employed for their e¡ects on tyro-sine phosphorylation of the cytoplasmic 66-kDa pro-tein and the peptide substrate PKS 1. It was ob-served that the known inhibitors of eukaryotickinases, such as genistein, staurosporine and dimeth-yl amino purine (DMAP), did not a¡ect the kinaseactivity of the cytoplasmic fraction of P. syringae asjudged by the ability of the cytoplasmic fraction tophosphorylate both the endogenous 66-kDa protein(Fig. 2) and the exogenous peptide substrate, PKS 1.All the above inhibitors have a common feature thatthey are known to compete with ATP for the nucleo-tide binding site of the tyrosine kinases [3,23,24]. Incontrast to these inhibitors, piceatannol which inhib-its Ptk competitively by binding to the protein-sub-strate binding site of the enzyme, was found to inhibitthe tyrosine kinase activity of P. syringae (Fig. 3).The half inhibitory concentration (IC50) was foundto be 150 WM which is much higher than the value(V15 WM) observed for eukaryotic enzymes [24].

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Fig. 2. The e¡ect of inhibitors on the phosphorylation of the cytoplasmic tyrosine-phosphorylated 66-kDa protein. The cytosolic extract(50 Wg of protein) from 22³C grown cells of P. syringae was phosphorylated with [Q-32P]ATP at 30³C for 10 min in the presence of kinaseinhibitors (genistein, staurosporine and dimethyl aminopurine) and phosphatase inhibitors (vanadate, sodium £uoride and okadaic acid)as described under Section 2. The proteins were separated by SDS-PAGE and processed for autoradiography. The tyrosine phosphoryl-ated 66-kDa protein is marked. The other phosphorylated marked proteins (85 and 30 kDa) are not phosphorylated at a tyrosine residueand have been described earlier [14,15]. Lanes 1^19 represent phosphorylation of proteins in the presence of 10, 5 and 1 mM dimethylaminopurine (lanes 1^3); 200, 100 and 50 mM sodium £uoride (lanes 4^6); 2, 1 and 0.5 WM okadaic acid (lanes 7^9); 100, 50 and 20 WMvanadate (lanes 10^12); 20, 10 and 1 WM staurosporine (lanes 13^15); 50 and 20 WM genistein (lanes 16 and 17) and without any inhibi-tor (control) (lanes 18 and 19).


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Fig. 4. The e¡ect of vanadate on the Ptk activity of P. syringae. The Ptk activities were estimated either by ELISA in the presence of thepeptide substrate PKS 1 (a) or by phosphorylation of the cytoplasmic 66-kDa protein in the presence of [Q-32P]ATP (b), as described inFig. 3. The values are the average of three experiments only. The protein contents for the cytosolic extracts in each assay were 75 Wg forELISA and 50 Wg for radioactive kinasing experiments.

Fig. 3. The e¡ect of piceatannol on the Ptk activity of P. syringae. The Ptk activities in the cytosol (100 Wg of proteins) were estimated ei-ther by ELISA in the presence of the peptide substrate PKS 1 (left panel) or by phosphorylation of the cytoplasmic 66-kDa protein inthe presence of [Q-32P]ATP (right panel). The Ptk activity in absence of the inhibitor was taken as 100% and the residual activity in thepresence of piceatannol has been plotted against the concentration of the inhibitor. The intensity of the 66-kDa protein bands in the ab-sence of the inhibitor on the Phosphoimager was taken as 100% and the residual intensity of the bands in the presence of various concen-trations of piceatannol has been plotted. The inset shows a representative image of the phosphorylated 66-kDa protein in the presence of0 (lane 1), 50 (lane 2), 150 (lane 3), 200 (lane 4) and 300 WM (lane 5) of piceatannol.


The kinase activity of P. syringae was also, sur-prisingly, found to be inhibited by sodium orthova-nadate, a known inhibitor of eukaryotic Ptpase [25].The IC50 of vanadate was 20 WM when the peptidesubstrate PKS 1 was used in the ELISA-based meth-od (Fig. 4a). However, the IC50 value for phospho-rylation of the cytoplasmic 66-kDa protein wasfound to be V3.5 WM (Fig. 4b), lesser than the valueobtained for phosphorylation of the exogenoussubstrate PKS1. This di¡erence could either be dueto the use of two di¡erent methods for the assay ordue to the di¡erence in the nature of the substrates.Among the other inhibitors which were tested, so-dium £uoride was found to inhibit the phosphoryla-tion of the 66-kDa protein but okadaic acid did notinhibit the Ptk activity. NaF inhibited the phospho-rylation of most of the cytosolic proteins, the reasonof which is not clear yet.

3.3. E¡ect of divalent cations on the Ptk activity ofP. syringae

The tyrosine phosphorylation of the 66-kDa pro-tein was found to be dependent on the divalent cat-ion Mg2� and optimal phosphorylation was ob-served at a concentration of 5 mM (Fig. 5). Theother divalent cations which were tested are Ca2�

and Mn2� (data not shown), both of which werealso e¡ective at 5 mM. At a higher concentrationof the above divalent cations, the phosphorylationof the 66-kDa protein dropped, but a new 62-kDaprotein was phosphorylated in the cytoplasm. Thenature of this protein is not known. A protein ofsimilar molecular mass was earlier found to be phos-phorylated more at a lower temperature (4³C) [15]. Itwas also observed that a new 20-kDa protein wasphosphorylated in the presence of Ca2� in the 80^100% ammonium sulfate fraction of the cytosol (Fig.5). The signi¢cance of this is also not clear yet.

3.4. Growth phase-dependent Ptk activity in thecytoplasm of P. syringae

In order to examine what could be a possible roleof the Ptk in the physiology of P. syringae, the levelof the kinase activity was measured in the cytosolicfractions which were isolated from di¡erent growthphases of the bacterium. The level of phosphoryla-

tion of the 66-kDa cytoplasmic protein increasedwith an increase in cell density (OD600) of the cultureand it reached a maximum at the stationary phase,both at low (4³C) and high (22³C) temperatures (Fig.6). The assay of the Ptk activity by ELISA in thepresence of exogenous substrate PKS 1 also indi-cated that the activity was the highest during thestationary phase of growth of P. syringae.

3.5. The e¡ect of vanadate on the growth ofP. syringae

Since the Ptk activity was found to be inhibitedby sodium orthovanadate, it was of interest to studythe e¡ects of the inhibitor on growth of P. syringaeat both low and high temperatures. P. syringae wasgrown at 4 and 22³C in ABM medium in the pres-ence of 125 and 250 WM sodium orthovanadate. At ahigh temperature (22³C), the inhibition on growthwas marginal. However, at a low temperature(4³C), there was a distinct increase in the lag periodof growth. The generation time of the bacteriumwas also a¡ected. For example, about a 3-fold in-crease in the generation time (from 6.5 to 21 h)

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Fig. 5. The e¡ect of metal cations on the phosphorylation of thecytosolic 66-kDa protein of P. syringae. The cytosolic proteins(50 Wg) were phosphorylated in the presence of [Q-32P]ATP as de-scribed under Section 2 and analysed by SDS-PAGE and autora-diographed. Phosphorylated protein bands have been shown inthe presence of 5, 10 and 50 mM CaCl2 (lanes 1^3) and MgCl2(lanes 4^6), respectively.


was noticed at 4³C in the presence of 250 WM vana-date in ABM medium (Fig. 7). Whether thegrowth inhibition in the presence of vanadate ob-served for P. syringae at 4³C is due to the lower

amount of Ptk activity in the cytoplasm or due tothe fact that Ptk functioning is vital for growthat this temperature remains to be solved. The possi-bility that the e¡ect of vanadate on growth is not

FEMSLE 8702 25-3-99

Fig. 7. The e¡ect of vanadate on growth of P. syringae at low (4³C) and high (22³C) temperatures. The cells were grown in ABM me-dium at low (left panel) and high temperatures (right panel) either in the absence (b), or in the presence of 125 WM (a) and 250 WM (E)sodium orthovanadate. The growth was followed by turbidity of the cultures at OD600.

Fig. 6. Levels of Ptk activity in the cytosol of P. syringae at various cell densities (OD600) during growth at low (4³C) and high (22³C)temperatures. The enzyme activity was measured at 30³C both by ELISA in the presence of the exogenous substrate PKS 1 (a) and by invitro phosphorylation of the endogenous 66-kDa protein (b). Activities at low temperature (-a-) and high temperature (-b-) grown cellshave been shown. The cytosol in each assay contained an equal amount of proteins, 75 Wg for ELISA and 50 Wg for 66 kDa phosphoryla-tion experiments, respectively.


related to the e¡ect of vanadate on the Ptk activityof P. syringae cannot be ruled out, since vanadateis also known to inhibit many ATPases, phospha-tases and the transport of phosphate into cells[26,27].

4. Discussion

Growth-related changes in the level of Ptk activityin P. syringae suggests that the so called `eukaryotickinase' may play an important role in the bacterialphysiology by post-biosynthetic modi¢cation of pro-teins. The activity of Ptk is at a maximum in P.syringae during the stationary phase of growthwhen there is normally a signi¢cant change in themetabolic activities of the cell. How much andwhat kind of role the Ptk of P. syringae might playin sensing and adjusting to the metabolic changeswould be of future interest.

The immediate interest was, however, to under-stand the biochemical similarities and di¡erences be-tween the Ptks of P. syringae and higher animalswhere the enzyme has undergone a wide diversi¢ca-tion, both structurally and functionally [28]. Fromthe present study it appears that the enzyme of P.syringae has a peptide-substrate binding site which isinhibited by piceatannol, but has an ATP bindingsite which is not inhibited by the kinase inhibitors,such as genistein, staurosporine and dimethyl aminopurine. Interestingly, none of the prokaryotic Ptksstudied so far was inhibited by genistein. The pro-karyotic Ptks, however, di¡er among themselves intheir substrate speci¢cities in vitro. The Ptk of P.syringae could not phosphorylate the synthetic sub-strate poly(Glu:Tyr), but could do so on peptidesubstrates, PKS 1 and PKS 2, corresponding to thetyrosine phosphorylation sites of eukaryotic proteinsp34cdc2 and gastrin, respectively. The Ptk of P. aeru-ginosa, on the other hand, could utilise poly(Glu:Tyr) as a substrate for tyrosine phosphorylation invitro [29,30]. In a recent study, however, a puri¢edmembrane-associated Ptk of A. johnsonii has beenshown to be incapable of phosphorylating syntheticpoly(Glu:Tyr) or angiotensin II as substrates [31].The enzyme was also not inhibited by genistein, sim-ilar to the enzyme of P. syringae. All these studiesindicate that there may be a divergence among the

prokaryotic Ptks which could be related to their bio-chemical properties.

The prokaryotic Ptks are also functionally diver-gent which is apparent from the location of the en-zymes and their cellular substrates. For example, the£agellar ¢lament protein of P. aeruginosa can bephosphorylated by Ptk which is located in the cellenvelope [29,30]. The Ptk activities of A. johnsoniiand P. solanacearum are also associated with thecell membrane [5,32]. The deduced amino acid se-quence of the gene for Ptk of A. johnsonii [31] showsstretches of amino acids and individual amino acidsat invariant positions which are generally consideredto be conserved for the eukaryotic protein kinasefamily [33]. Interestingly, however, the overall simi-larity of the enzyme lies within two bacterial pro-teins, EpsB of the phytopathogen P. solanacearumand ExoP of the plant symbiont Rhizobium meliloti.Both these proteins are involved in the productionand transport of exopolysaccharides [34,35]. In Nos-toc commune UTEX584, the 85-kDa tyrosine phos-phorylated protein is observed only in cells grown inmedia enriched in nitrogen [36]. In Myxococcus xan-thus, a multicellular prokaryote, the tyrosine phos-phorylated proteins are developmentally regulated[37]. In the psychrotrophic P. syringae, which hasbeen used in the present study, both the Ptk activityand the 66-kDa tyrosine-phosphorylated protein arelocated in the cytoplasm and the Ptk activity in-creases as the growth approaches the stationaryphase. Therefore, the Ptk of P. syringae might playan important role during the change of the metabolicstate of cells at the exponential to stationary phasetransition. A similar alteration of protein tyrosinephosphorylation was also observed in streptomycetes[38]. In the latter case however, the protein tyrosinephosphorylation decreased as the cells approachedthe stationary phase. In a more recent study, thetyrosine phosphorylated protein, Typ A (the productof o591) of E. coli has been shown to be involved inthe production of stress-related proteins, includingthe carbon starvation protein (Csp15) and universalstress protein (UspA) [6]. Therefore, the Ptks mightbe involved in regulation of the general metabolismof prokaryotes during various cellular stresses.

In conclusion, it appears that the Ptk of P. syrin-gae has some features common to eukaryotic Ptks,such as its ability to recognise eukaryotic peptide

FEMSLE 8702 25-3-99


substrates (PKS 1 and PKS 2) and in being inhibitedby piceatannol. But the enzyme also has similaritywith other prokaryotic Ptks as exhibited by genisteinresistance. However, the enzyme is unique in that itis inhibited by sodium orthovanadate which isknown to inhibit eukaryotic Ptpase. One of the plau-sible explanations of this inhibition is that the Ptk ofP. syringae is activated by the removal of a phos-phate which is brought about by a Ptpase in the cell.Consequently, in the presence of vanadate when thePtpase is inhibited, the Ptk of P. syringae remainsinactive and is unable to phosphorylate either theendogenous 66-kDa protein or the exogenous pep-tide substrates, such as PKS 1. This hypothesis ispartly supported by the fact that a Ptpase activitywhich is observed in the cytoplasm of P. syringae isalso inhibited by sodium orthovanadate (Jagtap andRay, unpublished observation). A clear picture, how-ever, would emerge only when we have the puri¢edenzymes (Ptk and Ptpase). The molecular character-isation of the enzymes and the encoding genes fromP. syringae would be of our major interest in thefuture.

Acknowledgments

We thank Dr. K. Uma Devi for her help duringthe estimation of the Ptk activity by the ELISAmethod and Dr. S. Shivaji for his constant interestin the study and useful comments on the manuscript.

References

[1] Kennelly, P.J. and Potts, M. (1996) Fancy meeting you here! afresh look at `prokaryotic' protein phosphorylation. J. Bacter-iol. 178, 4759^4764.

[2] Smith, S.C., Kennelly, P.J. and Potts, M. (1997) Protein ty-rosine phosphorylation in the Archaea. J. Bacteriol. 179,2418^2420.

[3] Hunter, T. and Cooper, J.A. (1985) Protein tyrosine kinases.Ann. Rev. Biochem. 54, 897^930.

[4] Farris, M., Grant, A., Richardson, T.B. and O'connor, C.D.(1998) BipA: a tyrosine phosphorylated GTPase that mediatesinteractions between enteropathogenic Escherichia coli(EPEC) and epithelial cells. Mol. Microbiol. 28, 265^279.

[5] Duclos, B., Grangeasse, C., Vaganay, E., Riberty, M. andCozzone, A.J. (1996) Autophosphorylation of a bacterial pro-tein at tyrosine. J. Mol. Biol. 259, 891^895.

[6] Freestone, P., Trinei, M., Clarke, S.C., Nystrom, T. and Nor-ris, V. (1998) Tyrosine phosphorylation in Escherichia coli.J. Mol. Biol. 279, 1045^1051.

[7] Ray, M.K., Seshu Kumar, G., Janiyani, K., Kannan, K.,Jagtap, P., Basu, M.K. and Shivaji, S. (1998) Adaptation tolow temperature and regulation of gene expression in Antarc-tic psychrotrophic bacteria. J. Biosci. 23, 423^435.

[8] Shivaji, S. and Ray, M.K. (1995) Survival strategies of psy-chrophilic bacteria and yeasts of Antarctica. Indian J. Micro-biol. 35, 263^281.

[9] Feller, G., Narnix, E., Arpigny, J.L., Aittaleb, M., Baise, E.,Genicot, S. and Gerday, C. (1996) Enzymes from psychrophil-lic organisms. FEMS Microbiol. Rev. 18, 189^202.

[10] Russell, N.J. (1998) Molecular Adaptations in psychrophilicbacteria: Potential for biotechnological applications. Adv.Biochem. Eng. Biotechnol. 61, 1^21.

[11] Stock, J.B., Ninfa, A.J. and Stock, A.M. (1989) Protein phos-phorylation and regulation of adaptive response in bacteria.Microbiol. Rev. 53, 450^490.

[12] Bourret, R.B., Borkovich, K.A. and Simon, M. (1991) Signaltransduction pathway involving protein phosphorylation inprokaryotes. Annu. Rev. Biochem. 60, 401^441.

[13] Parkinson, J.S. (1993) Signal transduction schemes of bacte-ria. Cell 72, 857^871.

[14] Ray, M.K., Seshu Kumar, G. and Shivaji, S. (1994) Phos-phorylation of membrane proteins in response to temperaturein an Antarctic Pseudomonas syringae. Microbiology 140,3217^3223.

[15] Ray, M.K., Seshu Kumar, G. and Shivaji, S. (1994) Tyrosinephosphorylation of a cytoplasmic protein from the Antarcticpsychrotrophic bacterium Pseudomonas syringae. FEMS Mi-crobiol. Lett. 122, 49^54.

[16] Shivaji, S., Shyamala Rao, N., Saisree, L., Sheth, V., Reddy,G.S.N. and Bhargava, P.M. (1989) Isolation and identi¢cationof Pseudomonas sp. from Schirmacher Oasis, Antarctica.Appl. Environ. Microbiol. 55, 767^770.

[17] Schnaitman, C.A. (1971) Solubilization of the cytoplasmicmembrane of Escherichia coli by Triton X-100. J. Bacteriol.108, 545^552.

[18] Laemmli, U.K. (1970) Cleavage of structural protein duringthe assembly of the head of bacteriophage T4. Nature 227,680^685.

[19] Cheng, H.-C, Nishio, H., Hatase, O., Ralph, S. and Wang,J.H. (1992) A synthetic peptide derived from p34cdc2 is a spe-ci¢c and e¤cient substrate of src family tyrosine kinases.J. Biol. Chem. 267, 9248^9256.

[20] Baldwin, G.S., Knesel, J. and Monckton, J.M. (1983) Phos-phorylation of gastrin-17 by epidermal growth factor stimu-lated tyrosine kinase. Nature 301, 435^437.

[21] Uma Devi, K., Jha, K. and Shivaji, S. (1998) A plasma mem-brane associated protein tyrosine phosphatase activity in ham-ster spermatozoa. Mol. Reprod. Dev. (in press).

[22] Markwell, M.K., Haas, S.M., Bieber, L.L. and Tolbert, N.E.(1978) A modi¢cation of the Lowry procedure to simplifyprotein determination in membrane and lipoprotein samples.Anal. Biochem. 87, 1057^1063.

[23] Munoz, G. and Marshall, S.H. (1990) An alternative method

FEMSLE 8702 25-3-99


for a fast separation of phosphotyrosine. Anal. Biochem. 190,233^237.

[24] Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Wata-nabe, S., Itoh, N., Shibuya, M. and Fukami, Y. (1987) Gen-istein, a speci¢c inhibitor of tyrosine speci¢c protein kinases.J. Biol. Chem. 262, 5592^5595.

[25] Geahlen, R.L. and McLaughlin, J.L. (1989) Piceatannol(3,4,3P,5P-tetrahydroxy-trans-stilbene) is a naturally occurringprotein tyrosine kinase inhibitor. Biochem. Biophys. Res.Commun. 165, 241^245.

[26] Huyer, G., Liu, S., Kelly, J., Mo¡at, J., Payette, P., Kennedy,B., Tsaprailis, G., Gresser, M.J. and Ramchandran, C. (1997)Mechanism of inhibition of protein tyrosine phosphatases byvanadate and pervanadate. J. Biol. Chem. 272, 843^851.

[27] Cantley, L.C., Jr., Cantley, L.G. and Josephson, L. (1978) Acharacterization of vanadate interactions with the (Na, K)-ATPase. J. Biol. Chem. 253, 7361^7368.

[28] Bowman, B.J., Allen, K.E. and Slayman, C.W. (1983) Vana-date-resistant mutants of Neurospora crassa are de¢cient inhigh a¤nity phosphate transport system. J. Bacteriol. 153,292^296.

[29] Suga, H., Kuma, K., Iwabe, N., Nikoh, N., Ono, K., Koya-nagi, M., Hoshiyama, D. and Miyata, T. (1997) Intermittentdivergence of the protein tyrosine kinase familiy during ani-mal evolution. FEBS Lett. 412, 540^546.

[30] South, S.L., Nichols, R. and Montie, T.C. (1994) Tyrosinekinase activity in Pseudomonas aeruginosa. Mol. Microbiol.12, 902^910.

[31] Kelly-Wintenberg, K., South, S.L. and Montie, T.C. (1993)Tyrosine phosphate in a- and b-type £agellus of Pseudomonasaeruginosa. J. Bacteriol. 175, 2458^2461.

[32] Grangeasse, C., Doublet, P., Vaganay, E., Vincent, C., Dele-age, G., Duclos, B. and Cozzone, A.J. (1997) Characterizationof a bacterial gene encoding an autophosphorylating proteintyrosine kinase. Gene 204, 259^265.

[33] Atkinson, M., Allen, C. and Sequeira, L. (1992) Tyrosinephosphorylation of a membrane protein from Pseudomonassolanacearum. J. Bacteriol. 174, 4356^4360.

[34] Hanks, S.K. and Quinn, A.M. (1991) Protein kinase catalyticdomain sequence database: identi¢cation of conserved fea-tures of primary structure and classi¢cation of family mem-bers. Meth. Enzymol. 200, 38^62.

[35] Huang, J. and Schell, M. (1995) Molecular characterization ofthe eps gene cluster of Pseudomonas solanacearum and its tran-scriptional regulation at a single promoter. Mol. Microbiol.16, 977^989.

[36] Becker, A., Niehaus, K. and Puhler, A. (1995) Low molecular-weight succinoglycan is predominantly produced by Rhi-zobium meliloti strains carrying a mutated ExoP protein char-acterised by a periplasmic N-terminal domain and a missingC-terminal domain. Mol. Microbiol. 16, 191^203.

[37] Potts, M., Sun, H., Mockaitis, K., Kennelly, P.J., Reed, D.and Tonks, N.K. (1993) A protein tyrosine/serine phosphataseencoded by the genome of the cyanobacterium Nostoc com-mune UTEX 584. J. Biol. Chem. 268, 7632^7635.

[38] Frasch, S.C. and Dworkin, M. (1996) Tyrosine phosphoryla-tion in Myxococcus xanthus, a multicellular prokaryote.J. Bacteriol. 178, 4084^4088.

[39] Waters, B., Vujaklija, D., Gold, M.R. and Davies, J. (1994)Protein phosphorylation in streptomycetes. FEMS Microbiol.Lett. 120, 187^190.

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Studies on the cytoplasmic protein tyrosine kinase activity of the Antarctic psychrotrophic bacterium Pseudomonas syringae